- Email: [email protected]
Effects of dietary vitamin E supplementation on spermatozoal quality in stallions with suboptimal post-thaw motility
Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
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Acknowledgements This work has been supported by Higher Agricultural School of Coimbra (ESAC), FCT (POCTI/CVT/49102/2002) and University of Coimbra (III/BIO/50/2005). doi:10.1016/j.anireprosci.2008.05.100 22 Effects of dietary vitamin E supplementation on spermatozoal quality in stallions with suboptimal post-thaw motility E.K. Gee a,∗ , J.E. Bruemmer a , P.D. Siciliano b , P.M. McCue a , E.L. Squires a a
Equine Reproduction Laboratory, Colorado State University, 3194 Rampart Road, Fort Collins, CO 80523-1693, USA b North Carolina State University, 220E Polk Hall, Raleigh, NC 27695-7621, USA E-mail address: [email protected] (E.K. Gee). Vitamin E is the major-lipid soluble antioxidant in cell membranes and plays an essential role as an inter- and intra-cellular antioxidant. Antioxidants in seminal plasma may protect spermatozoa against oxidative stress associated with reactive oxygen species, which may be induced by cooling and cryopreservation. The objective of this study was to determine the effects of vitamin E supplementation on sperm viability after cooling and freezing in stallions with poor post-thaw semen motility. Fifteen stallions of Quarter Horse, Arabian and Thoroughbred breeds, aged 7–26 years, were classified as having less than satisfactory semen freezing potential (post-thaw progressive motility ≤35%). Stallions were randomly assigned to one of two dietary treatments: control (CTRL, n = 7) or natural vitamin E supplementation (VITE, n = 8; D-alpha tocopherol; ADM Alliance Nutrition Inc) 3000 IU per day for 14 weeks. All stallions were fed mixed grass hay to maintain body condition scores of 4.5–5.5, and 0.5 kg of a pelleted ration (Safechoice, Nutrena) mixed with 20 ml soybean oil; VITE animals had vitamin E mixed with the pelleted ration and oil. Stallions were collected using an artificial vagina during weeks 0, 7 and 14. Two ‘cleanout’ collections were performed two days before semen was collected for analysis. Motility evaluations were computer assisted (Hamilton Thorn IVOS Motility Analyzer v12). Fresh semen was evaluated within 5 min of collection; cooled extended semen (E-Z Mixin CST, Animal Reproduction Systems) was evaluated at 24 and 48 h after warming at 37 ◦ C for 10 and 30 min. Two billion sperm cells were frozen using a lactose-based extender (E-Z Freezin LE). Post-thaw evaluations were carried out after warming at 37 ◦ C for 10 and 30 min. Data were analyzed by repeated measures with the PROC GLIMMIX procedure of SAS v9.1.3. The model contained treatment week, treatment, and week by treatment interactions. Stallion within treatment was a random effect. There were no significant treatment effects on total and progressive motility of raw semen, cooled semen at 24 h, or frozen–thawed semen. There were significant improvements in cooled semen of the VITE group compared to cooled semen of the CTRL group at 48 h evaluated after 10 and 30 min of warming (P = 0.01 for total motility at 10 min; P = 0.03 and P = 0.04 progressive motility at 10 and 30 min post-thaw, respectively). There were no significant differences in path velocity, straightness or percentage rapidly motile sperm due to treatment, with the exception of an improvement in percent rapidly motile cells 48 h after cooling with 10 min warming in VITE group compared to CTRL group (P = 0.02). These results indicate that dietary vitamin E
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Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
may have positive effects on spermatozoa motility after 48 h cooling, and further investigation is warranted. doi:10.1016/j.anireprosci.2008.05.101 23 Equine spermatozoa viability comparing the NucleoCounter SP-100 and the eosin–nigrosin stain C.S. Johansson a , F.C. Matsson a , H. Lehn-Jensen a , J.M. Nielsen b , M.M. Petersen a,∗ a Veterinary Reproduction and Obstetrics, Department of Large Animal Sciences, Faculty of Life Sciences, Copenhagen University, Denmark b Ansager Equine Hospital, Ansager, Denmark
E-mail address: [email protected] (M.M. Petersen). The objective of this study was to assess spermatozoal viability using eosin–nigrosin stain (E–N) and the NucleoCounter SP-100 (NC) and correlate viability to progressive motility and morphology. Spermatozoal viability or integrity of the sperm plasma membrane can be evaluated in several ways. Under field conditions, spermatozoal viability can be assessed using either classical stains, like E–N, or using the NC. When using the E–N stain, eosin is occluded from spermatozoa with an intact membrane, but penetrates membrane damaged cells. E–N is a subjective test using light microscopy and 100–200 spermatozoa are counted. The NC is an integrated fluorescence microscope that counts the number of spermatozoa (approximately 1500) stained with propidium iodide (PI). PI has been used extensively to stain non-viable spermatozoa (Love et al., 2003). Looking at boar semen the accuracy and repeatability of the NC was identical to the flow cytometer (Hansen et al., 2006). Further information on the NC at www.chemometec.dk. Spermatozoa viability using E–N and the NC as well as progressive motility and morphology were assessed in 72 ejaculates (8 ejaculates from 9 stallions) on a Danish stud farm at regular intervals throughout the breeding season 2007. Of the evaluated ejaculates, 3 were not included in the dataset due to suboptimal E–N slide preparation. Progressive motility was assessed immediately after collection in extended semen. Spermatozoal morphology was determined using the E–N stain (200 cells). In 67 of the 69 evaluated ejaculates the percentage of viable cells was higher when determined by E–N compared to the NC. The mean difference in spermatozoal viability determined by E–N and NC was 12.5%, but the difference was not significant (p = 0.09, two sample test for proportions). Spearman’s correlation coefficient (r) was 0.64 (p < 0.001). Sperm viability determined by E–N and NC correlated significantly to progressive motility and morphology: r = 0.72 and 0.57 and r = 0.79 and 0.60 (p < 0.001), respectively. The fact that viability in 67 of 69 ejaculates was higher when using E–N compared to the NC, could be explained by the fundamental difference in the two methods (eosin vs. PI). The subjectivity of E–N could very likely be a cause of variation since the difference between eosin stained or unstained spermatozoa is sometimes subtle.
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Acknowledgements This work has been supported by Higher Agricultural School of Coimbra (ESAC), FCT (POCTI/CVT/49102/2002) and University of Coimbra (III/BIO/50/2005). doi:10.1016/j.anireprosci.2008.05.100 22 Effects of dietary vitamin E supplementation on spermatozoal quality in stallions with suboptimal post-thaw motility E.K. Gee a,∗ , J.E. Bruemmer a , P.D. Siciliano b , P.M. McCue a , E.L. Squires a a
Equine Reproduction Laboratory, Colorado State University, 3194 Rampart Road, Fort Collins, CO 80523-1693, USA b North Carolina State University, 220E Polk Hall, Raleigh, NC 27695-7621, USA E-mail address: [email protected] (E.K. Gee). Vitamin E is the major-lipid soluble antioxidant in cell membranes and plays an essential role as an inter- and intra-cellular antioxidant. Antioxidants in seminal plasma may protect spermatozoa against oxidative stress associated with reactive oxygen species, which may be induced by cooling and cryopreservation. The objective of this study was to determine the effects of vitamin E supplementation on sperm viability after cooling and freezing in stallions with poor post-thaw semen motility. Fifteen stallions of Quarter Horse, Arabian and Thoroughbred breeds, aged 7–26 years, were classified as having less than satisfactory semen freezing potential (post-thaw progressive motility ≤35%). Stallions were randomly assigned to one of two dietary treatments: control (CTRL, n = 7) or natural vitamin E supplementation (VITE, n = 8; D-alpha tocopherol; ADM Alliance Nutrition Inc) 3000 IU per day for 14 weeks. All stallions were fed mixed grass hay to maintain body condition scores of 4.5–5.5, and 0.5 kg of a pelleted ration (Safechoice, Nutrena) mixed with 20 ml soybean oil; VITE animals had vitamin E mixed with the pelleted ration and oil. Stallions were collected using an artificial vagina during weeks 0, 7 and 14. Two ‘cleanout’ collections were performed two days before semen was collected for analysis. Motility evaluations were computer assisted (Hamilton Thorn IVOS Motility Analyzer v12). Fresh semen was evaluated within 5 min of collection; cooled extended semen (E-Z Mixin CST, Animal Reproduction Systems) was evaluated at 24 and 48 h after warming at 37 ◦ C for 10 and 30 min. Two billion sperm cells were frozen using a lactose-based extender (E-Z Freezin LE). Post-thaw evaluations were carried out after warming at 37 ◦ C for 10 and 30 min. Data were analyzed by repeated measures with the PROC GLIMMIX procedure of SAS v9.1.3. The model contained treatment week, treatment, and week by treatment interactions. Stallion within treatment was a random effect. There were no significant treatment effects on total and progressive motility of raw semen, cooled semen at 24 h, or frozen–thawed semen. There were significant improvements in cooled semen of the VITE group compared to cooled semen of the CTRL group at 48 h evaluated after 10 and 30 min of warming (P = 0.01 for total motility at 10 min; P = 0.03 and P = 0.04 progressive motility at 10 and 30 min post-thaw, respectively). There were no significant differences in path velocity, straightness or percentage rapidly motile sperm due to treatment, with the exception of an improvement in percent rapidly motile cells 48 h after cooling with 10 min warming in VITE group compared to CTRL group (P = 0.02). These results indicate that dietary vitamin E
ANIREP 3632 1–59
24
Abstracts / Animal Reproduction Science xxx (2008) xxx–xxx
may have positive effects on spermatozoa motility after 48 h cooling, and further investigation is warranted. doi:10.1016/j.anireprosci.2008.05.101 23 Equine spermatozoa viability comparing the NucleoCounter SP-100 and the eosin–nigrosin stain C.S. Johansson a , F.C. Matsson a , H. Lehn-Jensen a , J.M. Nielsen b , M.M. Petersen a,∗ a Veterinary Reproduction and Obstetrics, Department of Large Animal Sciences, Faculty of Life Sciences, Copenhagen University, Denmark b Ansager Equine Hospital, Ansager, Denmark
E-mail address: [email protected] (M.M. Petersen). The objective of this study was to assess spermatozoal viability using eosin–nigrosin stain (E–N) and the NucleoCounter SP-100 (NC) and correlate viability to progressive motility and morphology. Spermatozoal viability or integrity of the sperm plasma membrane can be evaluated in several ways. Under field conditions, spermatozoal viability can be assessed using either classical stains, like E–N, or using the NC. When using the E–N stain, eosin is occluded from spermatozoa with an intact membrane, but penetrates membrane damaged cells. E–N is a subjective test using light microscopy and 100–200 spermatozoa are counted. The NC is an integrated fluorescence microscope that counts the number of spermatozoa (approximately 1500) stained with propidium iodide (PI). PI has been used extensively to stain non-viable spermatozoa (Love et al., 2003). Looking at boar semen the accuracy and repeatability of the NC was identical to the flow cytometer (Hansen et al., 2006). Further information on the NC at www.chemometec.dk. Spermatozoa viability using E–N and the NC as well as progressive motility and morphology were assessed in 72 ejaculates (8 ejaculates from 9 stallions) on a Danish stud farm at regular intervals throughout the breeding season 2007. Of the evaluated ejaculates, 3 were not included in the dataset due to suboptimal E–N slide preparation. Progressive motility was assessed immediately after collection in extended semen. Spermatozoal morphology was determined using the E–N stain (200 cells). In 67 of the 69 evaluated ejaculates the percentage of viable cells was higher when determined by E–N compared to the NC. The mean difference in spermatozoal viability determined by E–N and NC was 12.5%, but the difference was not significant (p = 0.09, two sample test for proportions). Spearman’s correlation coefficient (r) was 0.64 (p < 0.001). Sperm viability determined by E–N and NC correlated significantly to progressive motility and morphology: r = 0.72 and 0.57 and r = 0.79 and 0.60 (p < 0.001), respectively. The fact that viability in 67 of 69 ejaculates was higher when using E–N compared to the NC, could be explained by the fundamental difference in the two methods (eosin vs. PI). The subjectivity of E–N could very likely be a cause of variation since the difference between eosin stained or unstained spermatozoa is sometimes subtle.
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